Biomedical subjects
Publications and source records attributed to M Nishimura.
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Proteoliposomes were reconstituted from detergent-solubilized pigment.protein complexes of chromatophores of Rhodopseudomonas sphaeroides and soybean phospholipids. The reconstituted vesicles showed a photooxidation of reaction center bacteriochlorophyll and a light-induced spectral shift of carotenoid to longer wave-lengths. The red shift similar to that in intact cells or chromatophores, indicates the generation of local fields in the membrane of proteoliposomes. When inside-positive membrane potential was induced by adding valinomycin and potassium salt, a shift of carotenoid spectrum to shorter wavelengths was observed. Therefore, the reconstituted vesicles, at least in the major part of population, produced the light-induced local field in the same direction as in intact cells, which is inside negative. Sidedness of the membrane structure and the direction of electric field formation in reconstituted vesicles were opposite to those in chromatophores (inside-out vesicles.
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Temperature-jump-induced absorbance changes of spinach chloroplasts in the dark were studied. After the temperature rise, a fast absorbance decrease and a succeeding slow absorbance increase were observed at the wavelength of 515 nm. The spectrum of the fast phase had positive maxima (increase in absorbance) at 430, 470 and 673 nm and a negative maxima (decrease in absorbance) at 525 nm. Permeant ions, tetraphenylboron-, tetraphenylarsonium+, and tetraphenylphosphonium+, decreased the extent of the fast absorbance change and increased the rate of slow recovery. Additions of inorganic potassium salts had a similar effect. Valinomycin, added in the presence of potassium ion, also increased the rate of slow recovery. These ions and ionophore had a parallel effect also on the recovery of flash-induced 515-nm absorbance change in chloroplasts. Electroneutral nigerericin did not affect the temperature-jump-induced absorbanc change. These results suggest the formation of electrical field across the thylakoid membrane in the dark accompanying the temperature rise. A possible involvement of the movement of water molecules (thermo-osmosis) in the observed absorbance changes is also discussed.
The shift of the carotenoid absorption spectrum induced by illumination and valinomycin-K+ addition was investigated in membrane structures with different characteristics and opposite sidednesses isolated from Rhodopseudomonas sphaeroides. Right-side-out membrane structures were prepared by isotonic lysozyme-EDTA treatment of the cells (spheroplasts) and by hypotonic treatment of spheroplasts (spheroplast membrane vesicles). Inside-out membrane structures ("chromatophores") were obtained by treating spheroplast membrane vesicles by French press or sonication. The membrane structures with either sidedness showed the same light-induced change of the "red shift" type. However, the absorbance change by K+ addition in the presence of valinomycin in the right-side-out membrane structures were opposite to that in the inverted vesicles, "blue shift" in the former and "red shift" in the latter. The carotenoid absorbance change was linear to membrane potential, calculated from the concentration of KCl added, with a reference on the cytoplasmic side, through positive and negative ranges.
Delayed fluorescence from bacteriochlorophyll in Chromatium vinosum chromatophores was studied at room temperature and under intermittent illuminations. The decay of delayed fluorescence was constituted of two components; a fast component decayed with a half time of about 8 ms, a slow one decayed in parallel with the reduction of photooxidized bacteriochlorophyll (P+) with a half time of 100-200 ms. The biphasic decay of delayed fluorescence indicated that a rapid equilibrium was established between the primary electron acceptor and the secondary acceptor. In the presence of o-phenanthroline, the time course of the decay of delayed fluorescence was identical with that of the reduction of P+ in reaction center-rich subchromatophore particles, although they did not necessarily coincide with each other in "intact" chromatophores. The intensity of the slow component was increased and the decay was accelerated at basic pH values. Reagents that dissipate the proton gradient across the chromatophore membranes such as carbonylcyanide m-chlorophenylhydrazone (CCCP) and nigericin accelerated the decay of the slow component. These effects are probably resulting from changes in internal pH of chromatophore vesicles. Reagents that dissipate the membrane potential such as CCCP and valinomycin decreased the intensity.
Delayed fluorescence from bacteriochlorophyll in the chromatophores of Chromatium vinosum, a photosynthetic purple sulfur bacterium, was studied in the presence of o-phenanthroline (o-phen) under intermittent illumination. Re-reduction of the photooxidized reaction center bacteriochlorophyll (P+) in the dark interval was accelerated by o-phen. This effect was attributed to the return of electrons trapped in the primary electron acceptor (A) to P+. In the presence of o-phen, the time course of the decay of delayed fluorescence was not coincident with that of the re-reduction of P+. The delayed fluorescence was somewhat intensified at the early stage (within 30 ms) of relaxation in the dark period. Prolonged illumination (longer than 20 ms) or uncouplers such as carbonylcyanide m-chlorophenylhydrazone (CCCP) or valinomycin plus nigericin decreased the intensity of delayed fluorescence and suppressed the stimulation of delayed fluorescence at the early stage. Delayed fluorescence from reaction center-rich subchromatophore particles decayed with a time course identical to that of the reduction of P+ and was not affected by CCCP, in the presence of o-phen. The intensification at the early stage in the chromatophores can be interpreted in terms of charge separation between pairs of P and A, primary electron donor and acceptor molecules, oriented perpendicular to the intact chromatophore membrane, the effect decreasing in parallel with the recombination of P+ and A-.
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Lysozyme-treated cells of a blue-green alga, Plectonema boryanum, had an internal pH of 7.3+/-0.2 under isotonic and hypotonic conditions. This value was similar to that of untreated cells. The CCCP-induced biphasic H+ change seen in the isotonic cells was not observed in the hypotonically treated cells. The biphasic time course remained in the hypotonic preparation if CaCl2 or MgCl2 was added prior to the osmotic shock. It is suggested that the cells have two compartments of H+ concentration. The outer region may be more acidic than the inner region. A light-induced H+ efflux was observed under isotonic conditions and an influx of H+ under hypotonic conditions. The H+ influx was not observed when lysozyme-treated cells were incubated with CaCl2 or MgCl2 prior to the hypotonic treatment. Two types of effects of divalent cations, one on the rigidity of the outer membrane and another on the permeability characteristics of the inner photosynthetic membrane, are indicated. Rearrangement of the photosynthetic membranes and an apparent inversion of the H+ pump by hypotonic shock are also suggested.
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Carbonylcyanide m-chlorophenylhydrazone (CCCP) or nigericin induced translocation of H+ In the dark across the cell membrane of blue-green algae Plectonema boryanum and Anacystis nidulans. The direction of the H+ flux depended on the pH of the suspending medium. At acidic pH, an influx of H+ and at alkaline pH an efflux of H+ were observed. It is suggested that the influx takes place at pH'S higher than the "internal" pH and the efflux at pH's lower than that. The internal pH was estimated to be 7.4+/-0.2 for Plectonema boryanum and 7.5+/-0.1 for Anacystis nidulans. Similar H+ changes due to CCP were observed under illumination, where the light induced efflux of H+ was limited by the counter-flux of cations. The internal pH of cells in the light, estimated from the pH-dependent reversion in the rate of the H+ change, was about 8.5.
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